12598 • The Journal of Neuroscience, December 14, 2016 • 36(50):12598–12610 Neurobiology of Disease Regulatory B Cells Induce Formation of IL-10-Expressing T Cells in Mice with Autoimmune Neuroinflammation X Andrea Pennati,1* Spencer Ng,2* Yuanqiang Wu,3 Jordan R. Murphy,2 XJiusheng Deng,2 X Srikant Rangaraju,4 Seneshaw Asress,4 XJennifer L. Blanchfield,5 Brian Evavold,5 and XJacques Galipeau1 1Department of Medicine, University of Wisconsin School of Medicine and Public Health and University of Wisconsin Carbone Cancer Center, Madison, Wisconsin 53706, Departments of 2Hematology and Medical Oncology, 3Neurology, and 4Microbiology and Immunology, Emory University, Atlanta, Georgia 30322, and 5Department of Oncology, Second Xiangya Hospital, Changsha 410011, Hunan, People’s Republic of China Although B cells are traditionally known for their role in propagating proinflammatory immune responses, their immunosuppressive effects have only recently begun to be appreciated. How these regulatory B cells (Bregs ) suppress the immune response remains to be ϩ worked out in detail. In this article, we show that Bregs can induce the formation of conventional FoxP3 regulatory T cells (Tregs ), as well ϩ ϩ as a more recently described CD49b CD223 regulatory T-cell subset, known as type 1 regulatory T cells (Tr1s). When Bregs are trans- ferred into mice with experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, they home to the spleen and mesenteric lymph nodes, leading to an expansion of Tregs and Tr1 in vivo.Tregs and Tr1s are also found in greater proportions in the CNS of mice with EAE treated with Bregs and are correlated with the remission of symptoms. The discovery that Bregs induce the formation of regulatory T-cell subsets in vivo may herald their use as immunosuppressive agents in adoptive cellular therapies for autoimmune pathologies. Significance Statement Although B cells are traditionally known for their role in propagating proinflammatory immune responses, their immunosup- pressive effects have only recently begun to be appreciated. How regulatory B cells (Bregs ) suppress the immune response remains to be fully understood. In this article, we show that Bregs can induce the formation of conventional regulatory T cells (Tregs ) as well as type 1 regulatory T cells (Tr1s). When Bregs are transferred into mice with experimental autoimmune encephalomyelitis (EAE), they home to secondary lymphoid organs, leading to an expansion of Tregs and Tr1s in vivo.Tregs and Tr1s are also found in greater proportions in the CNS of mice with EAE treated with Bregs and are correlated with the remission of symptoms. Introduction responses via the production of immunosuppressive cytok- ines, predominately interleukin (IL)-10, transforming growth Regulatory B cells (Bregs) have been ascribed important anti- inflammatory functions in a multitude of autoimmune condi- factor-␤ (TGF-␤), and, more recently, IL-35 (Tian et al., 2001; tions in both humans and mice, including colitis, arthritis, and Fillatreau et al., 2002; Shen et al., 2014; Rosser and Mauri, 2015). neuroinflammatory disorders (Mauri et al., 2003; Evans et al., A functional definition remains the current standard of classify- 2007; Blair et al., 2010; Ray et al., 2011; Yang et al., 2012). Bregs are ing Bregs due to a lack of consensus regarding subset-defining cell functionally defined by their ability to inhibit overt inflammatory surface marker expression or a lineage-specific transcription fac- tor (Rosser and Mauri, 2015; Tedder, 2015). The role that IL-10- Received June 22, 2016; revised Oct. 11, 2016; accepted Oct. 18, 2016. secreting Bregs play in disease has been most well documented in Authorcontributions:A.P.,S.N.,J.D.,S.R.,J.L.B.,andJ.G.designedresearch;A.P.,S.N.,Y.W.,J.R.M.,J.D.,S.R.,and experimental autoimmune encephalomyelitis (EAE), a murine S.A.performedresearch;A.P.,B.E.,andJ.G.contributedunpublishedreagents/analytictools;A.P.,S.N.,Y.W.,J.R.M., model of the human demyelinating disease, multiple sclerosis J.D., S.R., S.A., and J.G. analyzed data; A.P., S.N., S.R., and J.G. wrote the paper. (MS; Ray et al., 2014). Various groups, including our own, have This work was supported by National Institutes of Health Grant R01-AI-093881. National Multiple Sclerosis Society Grant FG1963A1/1 was awarded to J.L.B. shown that the adoptive transfer of Bregs into mice with EAE can *A.P. and S.N. contributed equally to this work. lead to durable remission of symptoms (Matsushita et al., 2008; The authors declare no competing financial interests. Rafei et al., 2009; Ray and Basu, 2014). However, the basic mech- Correspondence should be addressed to Dr. Jacques Galipeau, 1111 Highland Avenue, 3009 Wisconsin Institute anisms that underscore how B may induce EAE remission in for Medical Research, Madison, WI 53792. E-mail: [email protected]. regs DOI:10.1523/JNEUROSCI.1994-16.2016 vivo remain unknown. The fact that IL-10 and MHC-II have been Copyright © 2016 the authors 0270-6474/16/3612598-13$15.00/0 reported to be critical for Breg-mediated immunosuppression in • Pennati, Ng et al. GIFT15 Bregs Induce Treg and Tr1 Formation in EAE J. Neurosci., December 14, 2016 • 36(50):12598–12610 • 12599 EAE have steered the investigation toward interactions between harvested from culture, incubated with CD16/32 (Fc Block) antibodies ϩ Bregs and CD4 T cells, which initiate and drive EAE through for 30 min, followed by cell surface marker staining with fluorochrome- cognate antigen recognition by MHC-II and the pathologic se- conjugated antibodies for an additional 30 min at 4°C before flow cytom- cretion of IL-17 in a particular subset of these cells [T helper type etry analysis. Supernatant from untreated, and GMCSF-, IL-15-, and 17 (Th17) cells; Rafei et al., 2009; Yoshizaki et al., 2012]. GIFT15-treated B cells were analyzed for cytokine secretion (IL-10, IL-27, and TGF-␤, eBioscience; IL-35, BioLegend; and TNFSF18/ While B cells have traditionally been thought of as augment- ϩ glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related ing proinflammatory responses of CD4 T cells, Bregs have been protein (GITR) ligand, RayBiotech). reported to suppress interferon (IFN)-␥-secreting Th1 effector functions in favor of Th2-like responses (Lund and Randall, In vitro coculture ϩ 2010). Bregs have also been reported to dampen overt inflamma- B cells and CD4 T cells were isolated by negative selection (STEMCELL ϩ ϩ ϩ tion by inducing FoxP3 CD4 regulatory T cells (Tregs) in trans- Technologies) per manufacturer protocol. CD4 T cells were placed into plant models of islet allografts and collagen-induced arthritis coculture with freshly isolated splenic B cells (derived from C57BL/6 ϩ (Carter et al., 2011; Lee et al., 2014). Further, Bregs have been mice by negative selection) or GIFT15 Bregs at a ratio of 2:1. CD4 T cells shown to induce the formation of IL-10-secreting, FoxP3 Ϫ reg- were stimulated with anti-CD3/28 DynaBeads (ThermoFisher Scientific) ulatory CD4ϩ T cells, known as type 1 regulatory T cells (Tr1s), in and analyzed by flow cytometry for GFP/IL-10 expression 72 h postcul- mouse models of lupus and collagen-induced arthritis (Gray et ture. In MOG35–55 peptide restimulation cocultures, cells from the spleens or MLNs of Vert-X mice exhibiting symptoms of EAE (clinical al., 2007; Blair et al., 2010). However, Breg induction of regulatory score, 1–2) were placed into culture with B cells or GIFT15 Bregs in the T-cell function in EAE has not been demonstrated. ␮ presence of MOG35–55 peptide (10 g/ml; Sigma-Aldrich; see below for In an effort to elucidate the effect of Bregs on the induction of ϩ MOG35–55-induced EAE) at a ratio of 2:1. CD4 T cells were analyzed by regulatory T-cell responses in vivo, we sought to determine how flow cytometry for GFP/IL-10, CD25, FoxP3, CD49b, and CD223 ex- Breg treatment of mice with myelin oligodendrocyte glycoprotein pression 72 h postculture. ϩ (MOG)35–55-induced EAE alters the CD4 T-cell compartment in a green fluorescent protein (GFP)/IL-10 reporter mouse Western blot analysis model. Using a recombinant, bacterially derived fusion protein Cells were extracted in lysis buffer (Cell Signaling Technology) sup- consisting of granulocyte-macrophage colony-stimulating factor plemented with 1 mM phenylmethylsulfonyl fluoride and 1ϫ protease (GMCSF) and IL-15 (GIFT15; Rafei et al., 2009; Pennati et al., inhibitor (ThermoFisher Scientific). Samples were separated by 2014), we show that GIFT15 can convert splenic B cells into SDS-PAGE and immunoblotted for phospho-STAT3 (1:1000), phospho-STAT5 (1:1000), phospho-Akt (1:500), phospho-IKB (1: IL-10-secreting Bregs (GIFT15 Bregs). In addition to IL-10 secre- 500), phospho-p38 (1:500), phospho-JNK (1:500), phospho-Erk1/2 tion, we report for the first time that GIFT15 Bregs also secrete IL-27, a cytokine that is critical for the induction of Tr1s (Awasthi (1:500), STAT3 (1:2000), STAT5 (1:2000), and Erk1/2 (1:1000). All antibodies were obtained from Cell Signaling Technology. et al., 2007; Murugaiyan et al., 2009; Gagliani et al., 2013). In accordance with this observation, GIFT15 Bregs are capable not ϩ ϩ Adoptive transfer of GIFT15 Bregs or B cells only of inducing IL-10-producing CD25 FoxP3 Tregs, but also ϩ ϩ A donor mouse was killed, and the spleen was removed. Lymphocytes CD49b CD223 Tr1s, in an antigen-specific manner. GIFT15 were prepared as a single-cell suspension, and B cells were isolated with a Bregs home to the spleen and mesenteric lymph nodes (MLNs) STEMCELL Technologies kit. After culture for 4–5 d in complete R10 when adoptively transferred into mice and induce the remission media with the addition of recombinant GIFT15 (10 ng/ml), the cells of disease in mice with EAE.